Bacterial infections cause a tremendous burden to human health. One would be hard-pressed to avoid news about disease outbreaks, multi-drug resistant (MDR) bacteria, and the lack of new drugs in development pipelines. While some consider there to be a modest resurgence in antibiotic development, transmission of pathogenic bacteria is still a cause for great concern. The CDC estimates that one in every 20 hospitalizations will result in an acquired infection (Bragg et al., 2014, Infect. Dis. Nanomed., 808:1-13).
Bacterial biofilms cause perhaps even greater risks to health. Biofilms are established communities of bacteria that form a protective matrix composed of extracellular materials to defend the population against environmental threats (Fletcher et al., 2014, Tetrahedron 70:6373-6383). Due in part to inhibition of diffusion through this physical barrier (Bridier et al., 2011, Biofouling 27:1017-1032), antibiotic and antiseptic treatments can be 100-1000 times less effective against established biofilms. This directly affects settings that rely on routine treatments with disinfectants to prevent the spread of bacteria (i.e., hospitals, food manufacturing plants, residential settings). Biofilms are associated with over 80% of microbial infections (NIH Program Announcement for Research on Microbial Biofilms: http://grants.nih.gov/grants/guide/pa-files/PA-03-047.html), including periodontitis, endocarditis, and chronic lung infections such as those in cystic fibrosis (Goswami et al., 2014, ACS Applied Materials & Interfaces 6:16384-16394). Furthermore, biofilms are often associated with indwelling medical devices such as catheters and joint replacements. This results in tremendous health ramifications; hospital-acquired infections are estimated to affect up to 2M patients per year and ultimately cause up to 100,000 deaths per year in the United States alone (Bragg et al., 2014, Infect Dis. Nanomed. 1-13).
One of the most routine methods to combat bacteria is the use of quaternary ammonium compounds (QAC) in antiseptics (Goswami et al., 2014, ACS Applied Materials & Interfaces 6:16384-16394). QACs, including those in Lysol and Microban® formulations, are ubiquitous and have been employed for decades, being used for pre-operative hand-cleaning as early as 1935 (Domagk, 1935, Dtsch. Med. Wiss. 61:829-832, Noguchi et al., 2005, J. Med. Microbiol. 557-565); today, approximately 500,000 tons of QACs are used annually (Tezel and Pavlostathis, 2011, Role of Quaternary Ammonium Compounds On Antimicrobial Resistance In The Environment. In Antimicrobial Resistance in the Environment, First Edition. John Wiley & Sons, Inc, p 349). Quaternary ammonium compounds work by targeting and disrupting the barrier function of bacterial membranes, which leads to death of the microbe (Wimley, 2010, ACS Chem Biol 905-917). QACs are initially attracted to the predominantly anionic bacterial surface due to coulombic interactions. Structurally, there is significant similarity amongst these compounds, although direct activity comparisons of the antimicrobials against panels of bacteria (and MRSA in particular) are not readily available. Common QACs generally have a single cation, and a long-chained alkyl group, which provides a non-polar “arm” with which the bacterial membrane is disrupted (Hugo, 1967, J. Appl. Bacteriol. 30:17). While these QAC structures are regarded as reasonably non-toxic, as many can be directly applied to human skin or even used in oral therapies, it is now recognized that all of these structures are likely to be susceptible to bacterial resistance, and little antibiofilm activity is reported for these compounds.
An alarming trend that has garnered surprisingly little public attention is the diminishing effectiveness of mono- and bis-cationic QACs over time due to bacterial resistance (Bragg et al., 2014, Infect Dis. Nanomed. 1-13). Multiple genes that code for QAC resistance (such as qacA, qacB, and qacC, as well as the norA promoter) have been identified over the past decade (Bragg et al., 2014, Infect Dis. Nanomed. 1-13); these genes encode efflux pumps that can expel QACs. Such qac genes are found on easily transferrable plasmids that typically contain several other putative gene products, including teichoic acid translocation permease and various surface proteins designed to aid the bacterial cell in evading QACs (Jensen et al., 2010, Plasmid 64:135-42).
Over the past thirty years the identification of bacterial isolates with QAC resistance genes has risen dramatically (Jennings et al., 2015, ACS Inf. Dis. 1:288-303) and, as a result, there have been efforts to better understand the mechanisms by which antiseptics can lose efficacy (Schumacher et al., 2001, Science 294:2158-2163; Jennings et al., 2015, ACS Inf. Dis. 1:304-309). Resistance to traditional disinfectants such as benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC) has been identified in both Gram-positive and Gram-negative bacteria (Costa et al., 2013, Open Micro. J. 7:59-71; Poole, 2004, Clin. Microbiol. Infect. 10:12-26) and has presumably arisen through overuse and prolonged sub-lethal exposure. These compounds can in fact activate numerous resistance mechanisms, including physiological changes to bacterial cell membranes, as well as the production of transporter proteins, which efflux antibacterial agents (Poole, 2005, J. Antimicrob. Chemo. 56:20-51). More specifically, the qacAB/R system is one of the primary methods by which Gram-positive bacteria, specifically S. aureus, minimizes exposure to QAC compounds. Although QACs are lytic to cell membranes, they are capable of entering the cell at sub-MIC concentrations by passive diffusion. The compounds can then either be exported by the basal level of QacA (a transmembrane efflux pump) that is present, or bind with QacR, a negative transcriptional regulator of qacA. Following the binding of QAC compounds to the recognition site, QacR disassociates, allowing for the transcription of the gene, qacA. This leads to the increased production of QacA and the rapid efflux of the antimicrobial compounds from the cell. Other efflux proteins in Gram-positive bacteria include NorA (Jennings et al., 2015, ACS Inf. Dis. 1:288-303; Marchi et al., 2015, Microbiol. Res. 170:184-194); an analogous system has also been observed in Gram-negative bacteria through the efflux pumps AcrAB-TolC in E. coli (EC) and MexAB-OprM in P. aeruginosa (PA) (Costa et al., 2013, Open Micro. J. 7:59-71; Poole, 2004, Clin. Microbiol. Infect. 10:12-26; Holdsworth and Law, 2013, J. Antimicrob. Chemother. 68:831-839; Li et al., 1995, Antimicrob. Agents Chemother. 39:1948-1953).
It has been posited that efflux pumps are in fact multidrug transporters with alternate primary functions, having evolved to recognize and export a wide range of antibacterial and biocidal scaffolds (Schumacher et al., 2001, Science 294:2158-2163; Poole, 2005, J. Antimicrob. Chemo. 56:20-51). The evolutionary origins of some of these resistance mechanisms have been attributed to the recognition of natural product QACs such as berberine, sanguinarine, and chelerythrine produced by plants (Schumacher et al., 2001, Science 294:2158-2163; Jennings et al., 2015, ACS Inf. Dis. 1:304-309). This is evidenced by the crystal structure of berberine bound to QacR, which highlights the key electrostatic (acidic amino acid residues) and π-π (aromatic residues) interactions. Brennan et al. demonstrated that commercially available dyes—crystal violet and malachite green—fit neatly into the binding site for berberine; they noted, however, that this recognition motif was limited to mono- and biscationic QACs (Schumacher et al., 2001, Science 294:2158-2163).
Furthermore, QAC-resistance genes are often transferred with multidrug-resistance genes, further promoting the spread of these debilitating strains (Bragg et al., 2014, Infect Dis. Nanomed. 1-13; Noguchi et al., 2005, J. Med. Microbiol. 557-565; Buffet-Bataillon et al., 2012, Int J Antimicrob Agents 39:381-389; Zhang et al., 2011, J. Hosp. Infection 78:113-117; Muller et al., 2013, PLOS ONE 8:e76835; Raggi et al., 2013, Clinical Isolates. Clin Microbial 2:1000121). This can also be attributed to a plasmid containing multiple sets of resistance genes, shown to readily transfer in biofilms (Taitt et al., 2014, Antimicrob Agents Chemother. 58:767-781). And this association has been rising—the proportion of methicillin-resistant S. aureus (MRSA) strains bearing QAC resistance genes has increased sharply in a short period of time (Buffet-Bataillon et al., 2012, Int J Antimicrob Agents 39:381-389; Sidhu et al., 2002, Antimicrob. Agents Chemother. 46:2797). A review from the Sep. 12, 2014 issue of Science starkly announced: “The widespread use of biocides [which include quaternary ammonium compounds] coselects for antibiotic resistance genes and could promote the spread of multidrug resistance plasmids” (Laxminarayan, 2014, Science 345:1299-1301).
Amphiphiles—compounds with both polar and non-polar sections—represent one of the longest-serving and most effective classes of antimicrobial agents. Anionic amphiphiles have been protecting human health since the advent of soaps; exciting recent developments are represented by polyanionic dendritic structures (Meyers et al., 2008, J. Am. Chem. Soc. 130:14444-14445; Williams et al., 2007, J. Amtimicrob. Chemother. 59:451-458; Macri et al., 2009, Bioorg. Med. Chem. 17:3162-3168; Maisuria et al., 2011, Bioorg. Med. Chem. 19:2918-2926; Lu et al., 2013, Biomacromolecules 14:3589-3598). Cationic amphiphiles are likewise of great importance (Walker and Paulson, 2002, Quaternary Ammonium Compounds, Marcel Dekker, New York); while nearly every class of living organism employs cationic antimicrobial peptides in a host of defensive applications (Guani-Guerra et al., 2010, Clin. Immunol. 135:1-11), laboratory-derived quaternary ammonium compounds (QACs) have been used to defend human health for about a century (Jacobs, 1916, J. Exp. Med. 23:563-568; Jacobs, 1916, J. Exp. Med. 23:569-576; Jacobs, 1916, J. Exp. Med. 23:577-599; Domagk, 1935, Dtsch. Med. Wiss. 61:829-832).
QACs bearing long alkyl chains are classical examples of amphiphiles, displaying a variety of interesting physical properties, such as the capacity for micelle formation and gelation (Steichen, 2002, in Handbook of Applied Surface and Colloid Chemistry, ed. Holmberg, K. 310-347 John Wiley & Sons, Ltd., New York). QACs also enjoy extensive precedent and applications in bacterial cell membrane disruption, leading to their widespread use as antiseptics (Walker and Paulson, 2002, Quaternary Ammonium Compounds, Marcel Dekker, New York). Both synthetic QACs and peptide-based amphiphiles (notably, antimicrobial peptides or AMPs (Guani-Guerra et al., 2010, Clin. Immun. 135:1-11) are prevalent. However, aside from modified peptides, there are relatively few QACs in scaffolds of natural products.
Amongst the examples of natural products with permanent cationic charges based at nitrogen are a series of tetrahydroisoquinolinium structures isolated from the Chinese vine Gnetum montanum, including magnocurarine, cyclized derivatives thereof, and the latifolians (Rochfort et al., 2005, J. Nat. Prod. 68:1080-1082). Latifolian A demonstrated modest antimicrobial activity, with a MIC of 35 μm against Pseudomonas aeruginosa (Martin et al., 2011, J. Nat. Prod. 74:2425-2430). However, it only demonstrated 55% inhibition of methicillin-resistant Staphylococcus aureus (MRSA) at 350 μm while magnocuraine and its tetracyclic derivatives showed no effectiveness at this concentration, which perhaps correlates to the lack of an alkyl chain.
Related isoquinolinium structures bearing additional aromatic rings include chelerythrine, sanguinarine and berberine. Berberine, also identified from a Chinese herb, has shown micromolar activity against P. aeruginosa (Čerňáková, M. and Košt'álová, 2002, Folia Microbiol. 47:375-378). Other quinolinium natural products with a quaternary ammonium center include tabouensinium chloride (Wabo et al., 2005, Nat. Prod. Res. 19:591-595) and the quinocitrines (Kozlovsky et al., 2005, Appl. Biochem. Microbiol. 41:499-502). Finally, ageloxime D (Hertiani et al., 2010, Bioorg. Med. Chem. 18:1297-1311) and dehydroevodiamine (Park et al., 1996, Planta Med. 62:405-409) diversify this structural class and present a positive charge delocalized over two nitrogens.
While the preparation and testing of QAC-derived polymers has been pursued with increasing intensity over the past three decades (Panarin et al., 1971, Khim.-Farm. Zh. 5:24-28; Jaeger et al., 2010, Progress Polym. Sci. 35:511-577; Tahiro, 2001, Macromol. Mater. Eng. 286:63-87; Tew et al., 2010, Acc. Chem. Res. 43:30-39; Kenawy et al., 2007, Biomacromolecules 8:1359-1384; Mintzer et al., 2012, Mol. Pharmaceuticals 9:342-354; Munoz-Bonilla and Fernandez-Garcia, 2012, Prog. Polym. Sci. 37:281-339; Munoz-Bonilla et al., 2014, Polymeric Materials with Antimicrobial Activity: From Synthesis to Applications, RSC Publishing; Liu et al., 2015, J. Am. Chem. Soc. 137:2183-2186) the incorporation of multicationic QACs and corresponding antimicrobial testing of these polymers has received scarce attention; literature reports of multicationic QAC polymers often present little to no bioactivity or characterization data (Kenawy et al., 2002, J. Polym. Sci. Part Polym. Chem. 40:2348-2393; Dizman et al., 2004, J. Appl. Polym. Sci. 94:635-642; Ayfer et al., Des. Monomers Polym. 8:437-451; Gong et al., 2001, Sens. Actuators B 73:185-191). Results suggest that mono- and bis-QAC-derived polymers not only possess superior antimicrobial properties in comparison to their small molecular counterparts, but may also possess lower toxicity (Tahiro, 2001, Macromol. Mater. Eng. 286:63-87; Ganewatta et al., 2014, Chem. Sci. 5:2011-2016).
There is a continuing need in the art for novel antimicrobial agents with low toxicity profiles that also demonstrate activity against resistant bacterial strains. The present invention addresses this unmet need in the art.